Cytosine methylation serves as a critical epigenetic mark by modifying DNA-protein interactions that influence transcriptional states and cellular identity. 5-methylcytosine (5-mC) has generally been viewed as a stable covalent modification to DNA; however, the fact that 5-mC can be enzymatically modified to 5-hydroxymethylcytosine (5-hmC) by Tet family proteins through Fe(II) ?-KG-dependent hydroxylation gives a new perspective on the previously observed plasticity in 5-mC-dependent regulatory processes. Epigenetic plasticity in DNA methylation-related regulatory processes influences activity-dependent gene regulation, learning and memory, and repeat-associated transcript expression in the central nervous system (CNS). Hydroxylation of 5-mC to 5-hydroxymethylcytosine (5-hmC) presents a particularly intriguing epigenetic regulatory paradigm in the mammalian brain, where its dynamic regulation is critical. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically. Using this technology, we have generated genome-wide maps of 5-hmC in mouse cerebellum and hippocampus during development. Our analyses suggest dynamic regulation of 5-hmC during neurodevelopment. More specifically, we have identified both stable and dynamic DhMRs (Differential 5-hydroxymethylated regions) during neurodevelopment. We have also found that the overall abundance of 5-hmC is negatively correlated with the dosage of MeCP2, which is mutated in Rett syndrome. Intriguingly, loss of Mecp2 leads to the specific reduction of 5-hmC signals at dynamic DhMRs. These data together point to critical roles for 5-hmC-mediated epigenetic regulation in neurodevelopment and human diseases. In this proposed study, using the approach that we have established, we will examine the role of 5-hmC during neurodevelopment. Specifically, we plan to address the following aims: 1) To determine the genome-wide temporal and spatial distribution of 5-hmC during neurodevelopment. 2) To determine how the loss of Mecp2 alters genome-wide 5-hmC modification. 3) To determine the role of Tet proteins in learning and memory. The success of our planned work will define the fundamental role of 5-hmC in neurodevelopment as well as learning and memory.